1. General
The following publications provide conventional techniques of molecular biology. Such procedures are described, for example, in the following texts that are incorporated by reference:    1) Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratories, New York, Second Edition (1989), whole of Vols I, II, and III;    2) DNA Cloning: A Practical Approach, Vols. I and II (D. N. Glover, ed., 1985), IRL Press, Oxford, whole of text;    3) Oligonucleotide Synthesis: A Practical Approach (M. J. Gait, ed., 1984) IRL Press, Oxford, whole of text, and particularly the papers therein by Gait, pp 1-22; Atkinson et al., pp 35-81; Sproat et al., pp 83-115; and Wu et al., pp 135-151;    4) Animal Cell Culture: Practical Approach, Third Edition (John R. W. Masters, ed., 2000), ISBN 0199637970, whole of text;    5) J. F. Ramalho Ortigão, “The Chemistry of Peptide Synthesis” In: Knowledge database of Access to Virtual Laboratory website (Interactiva, Germany);    6) Sakakibara, D., Teichman, J., Lien, E. Land Fenichel, R. L. (1976). Biochem. Biophys. Res. Commun. 73 336-342    7) Merrifield, R. B. (1963). J. Am. Chem. Soc. 85, 2149-2154.    8) Barany, G. and Merrifield, R. B. (1979) in The Peptides (Gross, E. and Meienhofer, J. eds.), vol. 2, pp. 1-284, Academic Press, New York.    9) Bodanszky, M. (1984) Principles of Peptide Synthesis, Springer-Verlag, Heidelberg.    10) Bodanszky, M. & Bodanszky, A. (1984) The Practice of Peptide Synthesis, Springer-Verlag, Heidelberg.
2. Description of Related Art
Persistent back pain poses a significant economic burden to society, mainly in terms of the large number of work days lost by patients who develop chronic back pain. The major cause of persistent back pain is intervertebral disc (IVD) degeneration. In this respect, in USA alone approximately 5.7 million people are diagnosed with IVD degeneration each year.
Intervertebral Discs (IVDs)
An IVD is a specialized connective tissue composed of a pad of fibrocartilage found between the bony vertebrae of the spine. IVDs act as a shock absorber to cushion the compressive, rotational and tensile forces applied to the vertebral column. An IVD comprises at least three elements: a tough outer tissue called the annulus fibrosus (AF) comprising concentric layers of intertwined annular bands comprising primarily collagen type I fibers; a nucleus pulposus (NP) within the AF, comprising a viscous gel containing proteoglycan and water held loosely together by an irregular network of collagen type II and elastin fibers; and flat, circular plates of cartilage that connect the vertebrae above and below the disc to the AF. The major proteoglycan found in the NP is the glucosaminoglycan aggrecan which is high in chondroitin sulfate and keratin sulfate. This proteoglycan provides osmotic properties needed to resist compression in the disc (Adams and Roughley, Spine 31: 2151-2161, 2006). Cells of the NP are initially notochord cells that are gradually replaced during childhood by rounded cells resembling the chondrocytes of articular cartilage. Cells of the AF are fibroblast-like, elongated parallel to the collagen fibers in the AF. Cell density declines with age and is extremely low in adults, especially in the NP.
Fibrocartilage found in an IVD differs to other forms of cartilage, e.g., hyaline cartilage or elastic cartilage. For example, the fibrocartilage found in IVDs contain cartilage-1 or type-1 cartilage, whereas this form of cartilage does not occur in hyaline cartilage or elastic cartilage. Moreover, the extracellular matrix within an IVD differs from that found in other cartilage, e.g. hyaline cartilage, in so far as it contains a high proteoglycan to collagen ratio, e.g., extracellular matrix of IVD has a ratio of proteoglycan to collagen of about 27:1, whereas hyaline cartilage has a ratio of about 2:1 (Mwale et al., European Cells and Materials, 8: 58-64, 2004). The increased level of proteoglycan relative to collagen in an IVD explains to some degree the gelatinous nature of an IVD, which is required for transmitting load applied to the IVD and providing the shock absorbing nature of these organs. In contrast to IVD, other forms of cartilage, e.g., hyaline cartilage or articular cartilage operate in isolation and must retain their own shape and, as a consequence, a higher concentration of collagen to proteoglycan is desired to provide such a firm and resilient nature (Mwale et al., supra).
At the microscopic level proteoglycans of IVD extracellular matrix also differ from those of other forms of cartilage, including articular cartilage, nasal cartilage, growth plate cartilage and menesci. For example, articular cartilage nasal cartilage, growth plate cartilage and menesci contain large aggregates of proteoglycan formed from hyaluronic acid central filaments in addition to large nonaggregated monomers. In contrast to these cartilages, IVDs contain short non-aggregated proteoglycan monomers and clusters of monomers without central filaments (Buckwalter et al., J. Orthop. Res., 7: 146-151, 1989). These differences in composition of IVDs and other forms of cartilage are indicative of significant differences in collagen and/or proteoglycan metabolism between these tissues.
IVD degeneration is associated with a series of biochemical and morphologic changes that combine to alter the biomechanical properties of the disc. During IVD degeneration, the concentration of proteoglycans in the NP and the water retaining potential of the disc decrease dramatically. There are also changes in the collagen content of the NP as the synthesis of type II collagen declines and the synthesis of less tensile type I collagen increases. Another change is a shift in phenotype of the differentiated chondrocyte of the NP into a more fibrotic type.
IVD Development
In adult life, events unfolding as a consequence of injury to the disc may mimic some of the molecular events that control the development of the disc. Development of the disc is under tight molecular control both temporally and spatially. Notochordal cells are involved in the development of the spinal cord and vertebra and they also contribute towards the patterning and differentiation of the IVDs. During gastrulation, the axial mesoderm gives rise to the notochord and somites develop into two parts: a schlerotome and a dermomyotome. The cells of the schlerotome are responsible for the formation of the spine and the IVD as the schlerotomes migrate toward and around the notochord and neural tube, and later separate into areas of loosely packed cells which go on to form the NP and a densely packed cells which form the AF.
IVD Related Disorders
Kippel Feil Syndrome (KFS) is a congenital condition characterized by the fusion of two or more cervical vertebrae (Type I-III; Kaplan et al., The Spine Journal 2005 5:564-576). This abnormality is the result of a failure of proper segmentation of vertebrae in the cervical region during embryonic development (Clark et al., 1998, Pediatr Radiol 28:967-974). In KFS the IVD(s) are not developed (hypo/oligogenesis) or there is an agenesis of the disc(s). Notwithstanding that a number of de novo PAXJ missense mutations, as well as PAX1 haploinsufficiency, i.e., reduced expression of PAXJ, have been associated with KFS, no definitive genetic basis for KFS has yet been identified.
Fibrodysplasia ossificans progressive (FOP) is a rare autosomal dominant disorder of connective tissue whereby patients also present with cervical spine abnormalities. FOP, a condition where there is excessive bone formation is often misdiagnosed for KFS, which has been identified by the present inventor as being an hypo/oligogenesis of the disc. Knockout mice which do not express the bone morphogenetic protein (BMP) antagonist noggin, exhibit a phenotype almost identical to FOP patients. Whilst the noggin gene (NOG) is not mutated in FOP, overactivity of the BMP pathway (i.e., enhanced BMP signaling) has been suggested as the molecular pathogenesis of FOP (e.g., in incorrect development of IVDs) (Schaffer et al., Spine 2005 30 (12): 1379-1385).
Bone Morphogenetic Proteins
BMPs are low-molecular weight glycoproteins that control many developmental processes. BMPs are multi-functional growth factors that belong to a larger family of related secreted factors, the transforming growth factor (TGF)-β superfamily. To date, around 20 BMP family members have been identified and characterized. Members of the BMP family include, for example, BMP-2, BMP-4, BMP-5, BMP-6, the osteogenic proteins OP-1 (BMP-7) and OP-2 (BMP-8), osteogenin (BMP-3), and BMP-9 to BMP-12. Other names for BMPs include growth and differentiation factors (GDF) and cartilage-derived morphogenetic proteins, e.g., CDMP-1 and -2, also known as GDF-5 and GDF-6/GDF-6, respectively. Notwithstanding that BMPs were first identified by virtue of their ability to promote ectopic cartilage and bone formation, BMP signaling plays a critical role in heart, limb, kidney, and skeletal development, and control many key steps in the formation and differentiation of the vertebrate nervous system.
BMPs signal through a molecular pathway, which is initiated by contact of extracellular BMPs with a high-affinity complex of heteromeric type II and type I serine/threonine kinase receptors. The receptor complexes in turn phosphorylate receptor regulated R-Smads 1, 5 and 8 which induces them to bind Smad4 (Co-Smad) and accumulate in the nucleus where they regulate transcription. The heteromeric BMP-regulated Smad complex can bind directly, or through other transcriptional partners to BMP response elements of gene promoters of xVent2, xVent2B, Msx1, Msx2, Hex, Smad7, and Id1. The pathway is further controlled by the action of inhibitory Smads 6 and 7 and by soluble antagonists that bind extracellular BMPs inhibiting binding to heteromeric complexes such as, Noggin, Chordin and Dan.
The manner in which BMPs regulate such diverse processes is largely determined by the cellular and tissue context in which the BMP signals are received. For example, although the molecular components of BMP signaling may be highly conserved, tissue and cell-type specificity ultimately determine which BMP and combinations of receptors, intracellular mediators, and extracellular antagonists control a particular process. BMP-regulated gene expression is further controlled by interaction of Smads with tissue-specific transcription factors and cross-talk with other signalling pathways to mediate the diverse transcriptional programs associated with BMP regulated processes.
Notwithstanding our increased understanding of the molecular events involved in development of an IVD, this understanding has yet to lead to the development of an effective treatment for a spinal disorder and/or spinal pain. Rather current treatment options for a spinal disorder and/or spinal pain require surgical intervention to replace a degenerated IVD and/or remove the IVD and fuse vertebrae. In this respect, spinal fusion is expensive because it requires prolonged hospitalisation and specialist surgical expertise. Furthermore, studies suggest that in the long-term, spinal fusion actually promotes degeneration at sites adjacent to the lumbar fusion. Furthermore, replacement of the disc is a major operation and despite potential benefits, many sufferers of repeated chronic neck pain and/or back pain avoid major spinal reconstruction. It is clear from the foregoing that there remains a need for compositions and methods for the treatment of spinal disorders and/or spinal pain, e.g. a spinal disorder associated with IVD degeneration, that does not require a prolonged period of hospitalization and/or that does not aggravate the spinal disorder and/or spinal pain. Ideally, this treatment should have the potential of regenerating disc tissue and/or preventing or slowing spinal degeneration.